The increasingly frequent outbreaks of infections by West Nile Virus (WNV) and other related flaviviruses in humans, as well as their recent categorization by the Centers for Disease Control and Prevention (CDC) as potential bioterrorism pathogens, have underscored the global public health need for effective antiviral chemotherapeutics and vaccines. Flavivirus infections are a global public health problem with about half of the flaviviruses causing human diseases (C. G. Hayes, in The Arboviruses: Epidemiology and Ecology, T. P. Monathy, ed., CRC, Boca Raton, Fla., vol. 5, chap. 49 (1989); M. J. Cardosa, Br Med Bull, 54, pp. 395-405 (1998); Z. Hubalek and J. Halouzka, Emerg Infect Dis, 5, pp. 643-50 (1999)). These viruses are normally maintained in a natural cycle between mosquito vectors and birds, whereas humans and equine are considered dead-end hosts. Birds, including the American crow, Corvus brachyrhynchos, can serve as non-human reservoirs for the virus. In the case of WNV, the viruses are transmitted to man by mosquitoes and in the Northeastern United States these mosquito vectors are primarily of the genera Culex and Aedes, in particular C. pipiens and A. vexans. 
West Nile virus and other related flaviviruses are classified within the family Flaviviridae, genus Flavivirus, and belong to the Japanese Encephalitis antigenic complex of viruses. In addition to WNV, this flavivirus sero-complex includes Japanese encephalitis virus (JEV), St. Louis encephalitis (SLEV), Alfuy virus (AV), Koutango virus (KV), Kunjin virus (JV), Cacipacore virus (CV), Yaounde virus (YV), and Murray Valley encephalitis virus (MVEV). The Flaviviridae family also includes the Tick-borne encephalitis virus (TBEV), Dengue virus (including the four serotypes of: DENV-1, DENV-2, DENV-3, and DENV-4), and the family prototype, Yellow Fever virus (YFV).
Flaviviruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality. A combined toll of hundreds of millions of infections around the world annually coupled with the lack of sustained mosquito control measures, has distributed flaviviruses throughout the tropics, subtropics, and temperate areas. As a result, over half the world's population is at risk for flaviviral infection. Further, modern jet travel and human migration have raised the potential for global spread of these pathogens.
WNV was originally isolated in 1937 from the blood of a febrile woman from Uganda's West Nile province and was subsequently found in many regions, including Africa, the Middle East, Europe, Russia, India and Indonesia. Most recently, WNV has appeared in North America, beginning with a 1999 outbreak in the New York City area.
Since 1996, WNV outbreaks have frequently occurred in humans and horses, including in Romania and Morocco in 1996; Tunisia in 1997; Italy in 1998; Israel, Russia and the U.S. in 1999; Israel, France and the U.S. in 2000; and Israel and the U.S. in 2001. Severe human disease associated with WNV infection has been reported worldwide, with 393 cases in 1996 in Romania; 942 cases in 1999 in Volgograd, Russia; and 2,417 and 45 cases in 1999 in Israel in 2000 and 2001, respectively. Since its appearance in northeastern U.S. in 1999, WNV has caused significant human, equine and avian disease, and has quickly spread from the Northeast to the eastern seaboard, to the Midwest, and most recently to the Deep South. There were 61 human cases (7 deaths) in New York City in 1999; 21 human cases (4 deaths) in New York, New Jersey and Connecticut in 2000; and 48 human cases (5 deaths) in New York, Florida, New Jersey, Connecticut, Maryland, Massachusetts, Georgia and Louisiana in 2001. As of Sep. 13, 2002, 1438 human cases (64 deaths) for year 2002 have been reported in over 30 states in the U.S. These data clearly indicate that human outbreaks of WNV pose a severe threat to public health.
Strains of WNV are categorized into two different phylogenetic lineages, namely, lineage I and II, which share 75% nucleotide sequence identity (Lanciotti, R et al, (2002) Virology 298:96-105). Lineage I strains have been isolated from human and equine epidemic outbreaks from around the world and constitute the main form of human pathogen. Sequence analysis indicates that the current epidemic strain in North America belongs to lineage I. Lineage II strains are rarely isolated from humans and are geographically restricted primarily to sub-Saharan Africa and Madagascar. The differences in disease patterns of lineage I and II strains are postulated to be the result of differences in vector competence (host compatibility), virulence, and transmission cycles of the strains, as well as, host immunity (Beasley, D. W. C. et al, (2001) International Conference on the West Nile Virus, New York Academy of Science Poster Section 1:5). Sequence analysis showed that the strain in North America is closely related to other human epidemic strains isolated from Israel, Romania, Russia, and France, all of which belong to lineage I (Lanciotti, R. et al. (1999) Science 286:2333-2337).
The flavivirus genome, including the genome of WNV, is a single positive-sense RNA of approximately 10,500 nucleotides containing short 5′ and 3′ untranslated regions (UTR), a single long open reading frame (ORF), a 5′ cap region, and a non-polyadenylated 3′ terminus. The entire genome is transcribed as a single polycistronic messenger RNA molecule, which is then translated as a polyprotein. Individual proteins are subsequently produced by proteolytic processing of the polyprotein, which is directed by viral and host cell proteases (Chambers, T. J. et al, (1990) Ann. Rev. Microbiol. 44: 649-688; Lindenbach, B. D. and C. M. Rice, (2001) In D. M. Knipe and P. M. Howley (ed), Fields virology, 4th ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, Pa.).
During the replication cycle of flaviviruses, especially WNV, synthesis of positive and negative (hereafter referred to as plus (+) and minus (−), respectively) sense RNAs is asymmetric. In the case of WNV, plus-sense RNAs are produced in 10- to 100-fold excess over minus-sense RNA. Regulatory sequences in the 3′ UTR are believed to function as a promoter for initiation of minus-strand RNA synthesis. Deletion of this region ablates viral infectivity (Brinton, M. A. et al, (1986) Virology 162: 290-299; Proutski, V., et al (1997) Nucleic Acids Res. 25: 1194-1202; Rauscher, S., et al (1997) RNA 3: 779-791).
With respect to the flavivirus genome, two distinct classes of genes are found which encode either structural or non-structural proteins. There are three structural proteins, which include capsid (C), membrane (M) or premembrane (prM), and envelope (E) glycoprotein. The E glycoprotein and M proteins are found on the surface of the virion where they are anchored in the membrane. Mature E glycoprotein is glycosylated, whereas M is not, although its precursor, prM, is a glycoprotein. In addition, there are seven non-structural proteins, which are denoted as NS1 (non-structural protein 1), NS2A, NS2B, NS3, NS4A, NS4B, and NS5. NS1 and NS2A correspond to a glycoprotein, NS2B corresponds to a protease cofactor, NS3, NS4A, and NS4B correspond to a protease and a helicase, and NS5 corresponds to a viral RNA-dependent RNA polymerase. In addition to these ten genes, regulatory elements in the 5′ and 3′ UTRs are also required for proper replication and packaging of the virions. The 5′ and 3′ UTRs, which are approximately 100 and 400-700 nucleotides in length, respectively, form highly conserved secondary and tertiary structures, conferring specificity in binding of host proteins such as the eukaryotic translation elongation factor, eF1-α (Blackwell, J. L., and M. A. Brinton, (1995) J. Virol. 69: 5650-5658; Blackwell, J. L., and M. A. Brinton, (1997) J. Virol. 71: 6433-6444).
The current global risk for flaviviral infections, the rapid and recent spread of WNV in the Western Hemisphere, and the recent CDC prioritization of WNV, together with DENV, JEV and YFV, as potential bioterrorism pathogens, highly stresses the need for anti-flaviviral treatments, such as vaccines and anti-flaviviral inhibitors. With the exception of a limited number of vaccines available for specific flavivirus infections, such as vaccines for YFV, TBEV, and JEV, effective vaccines and/or anti-flaviviral therapies for use in humans are not generally available for flavivirus infections, especially WNV and DENV. Thus, new effective vaccines and/or anti-flaviviral therapies for the immunization and/or treatment of flavivirus infections, especially WNV and DENV, in humans are urgently needed and would be an advance in the art.
Specifically, in the case of WNV, there are no vaccines or effective anti-WNV therapies, inhibitors, medications, or cures available for the immunization against or the treatment of WNV infections. To prevent WNV infection in humans, mosquito control and public awareness programs aimed at reducing exposure to mosquito bites are often deployed. The efficacy and the cost-effectiveness of these prevention measures are, however, compromised by the sporadic nature of human WNV epidemics. Further, mosquito control programs, such as, insecticide spraying, are difficult, potentially toxic to humans, and require repeated application. Further, insecticide spraying does not provide complete coverage of mosquito breeding areas or eradiction of mosquitoes.
Currently, the only way of treating WNV infection is to treat the symptoms of infection. Symptoms are highly variable and generally occur 5-15 days following the bite of an infected mosquito. Some infected persons may have no noticeable symptoms or may experience only mild illness, such as a slight fever, headache, rash, swollen nodes and conjunctivitis, before fully recovering. However, in other infected persons, particularly the elderly, WNV can cause serious disease that may include a rapid onset of severe headache, high fever, stiff neck, disorientation, and muscle weakness. At the most severe level, WNV infection can cause permanent neurological damage, such as with encephalitis or meningitis, and can be fatal. Treatment is supportive, often involving hospitalization, intravenous fluids, respiratory support, and prevention of secondary infections for patients with severe disease.
Similarly, in the case of DENV, there are no effective vaccines and/or antiviral therapies available. Over 2.5 billion people worldwide live in areas at risk of DENV infection, and 100 million people are affected annually. Classic dengue fever is characterized by acute onset of high fever, frontal headache, retro-orbital pain, nausea, vomiting, and often a maculopapular rash. In addition, many patients may notice a change in taste sensation. Symptoms tend to be milder in children than in adults, and the illness may be clinically indistinguishable from influenza, measles, or rubella. The disease manifestations can range in intensity from inapparent illness to the symptoms described. The acute phase of up to 1 week is followed by a 1- to 2-week period of convalescence which is characterized by weakness, malaise, and anorexia. In leiu of a vaccine and/or anti-DENV chemotherapeutics, the only available treatments emphasize relief of the disease symptoms and can include fluid replacement and acetaminophen administration. Development of a DENV vaccine has long been unsuccessful principally because of the need to simultaneously immunize and induce long-lasting protection against all four DENV serotypes (DENV-1, -2, -3, and -4). An incompletely immunized individual may be sensitized to dengue hemorrhagic fever or dengue shock syndrome.
As outlined above, effective chemotherapeutics to treat WNV and DENV and other emerging flaviviruses are urgently needed. Although a limited number of inhibitors of flaviviruses have been identified, many of these have severe side effects, are not specific to flaviviruses, and are not known to be clinically effective and/or useful. For example, recent evidence suggested the use of nucleoside analogs as potential inhibitors of flaviviruses. Specific examples include inhibitors of orotidine monophosphate decarboxylase, inosine monophosphate dehydrogenase, and CTP synthetase. Although it appeared that these inhibitors may have been effective in virus infected Vero cells, their effectiveness in humans or animals (i.e., in vivo) is not known. Additionally, as these nucleoside analogs are broad-spectrum inhibitors of purine and pyrimidine biosynthesis, the occurrence of side effects and lack of flaviviral specificity would further limit their usefulness in a clinical setting.
Another nucleoside analog, the drug Ribavirin, was found to have some activity against WNV in vitro when administered in combination with interferon alpha-2b. However, the drug combination has not been shown to be effective in humans. Similarly, inhibitors to other protein activities of the viral genome, such as the helicase and protease activities encoded by NS3, have been explored; however, their clinical significance is unknown since their anti-WNV activities have not been tested in vivo. Finally, inhibitors of viral glycoprotein processing have been studied, but the prevalence of side effects due to inhibition of N-linked glycosylation, as well as difficulty in achieving therapeutic serum concentration levels, limit the usefulness of this type of compound. Thus, although there are a small number of known inhibitors for flaviviruses, none have been shown to be effective in humans. Accordingly, novel anti-flavivirus chemotherapies and/or improvements in the effectiveness, specificity, and clinical utility of known flavivirus chemotherapies, would be an advance in the art.
As detailed above, the development of therapeutic drugs and/or vaccines to treat and/or immunize against WNV, DENV and other emerging flavivirus infections is urgently needed and of great importance to global public health. To achieve this goal, it is essential to develop high-throughput screening assays to facilitate the identification of novel chemotherapeutics effective against flaviviruses or vaccines capable of establishing a protective immune response to flaviviruses. Two general strategies to be adapted for the screening and identification of novel chemotherapeutic antiflaviviral compounds and/or vaccines are based on biochemical and genetic approaches.
Assays for screening antiviral compounds that are based on biochemical approaches typically involve testing compounds for activities that limit or inhibit viral enzymes or proteins that are essential for viral propagation. For example, NS3, which has protease, helicase and NTPase activity, and NS5, which has an RNA-dependent RNA polymerase and methyltransferase activity, are key components of viral replication complex and thus, are ideal targets for antiviral screening. Further, three-dimensional structures of viral proteins, if available, can afford the possibility for rational design of drugs that will inhibit their activity, i.e., designing drugs based on the knowledge of the structure and shape of the active sites of the protein. For example, the crystal structures of the DENV NS3 protease domain and NS5 cap methyltransferase fragment have been solved and thus, the possibility of rationally designing small molecules to inhibit the active sites of NS3 and NS5 is feasible. Although biochemical approaches are capable of identifying potential viral inhibitors, they are limited in their overall efficiency since only a single enzyme or protein can be tested for any potential assay. Thus, individual assays would be required to screen for inhibitors of each given viral target protein.
In contrast, assays utilizing a genetic approach, which are usually cell-based, offer a number of advantages over biochemical approaches. One major advantage of a genetic approach based assay is that multiple viral protein targets can be analyzed simultaneously. A second major advantage is that, since genetic assays involve the use of living cells and the uptake of compounds therein, the screening assay is administered in a more authentic therapeutic environment. Accordingly, inhibitors identified through cell-based assays typically have a higher success rate in subsequent animal experiments.
Currently, there is only one type of cell-based assay available for screening for flaviviral inhibitors. The assay involves the infection of cultured cells with virus and the subsequent monitoring for potential inhibition in the presence of a potential inhibitor through observation or quantification of cytopathic effects (J. D. Morrey et al., Antiviral Res (2002) 55:107-116; I. Jordan, J. Infect. Dis. (2000) 182:1214-1217) or quantification of viral RNA by reverse transcriptase (RT)-PCR(S. F. Wu, J. Virol. (2002) 76:3596-3604). These assays are highly labor-intensive and impossible to use when screening compound libraries in large quantities. Thus, there is an urgent need in the art to develop reliable, high-throughput cell-based assays for the rapid screening and identification of potential inhibitors from compound libraries. Towards this endeavor, “reverse genetics systems”, which are cDNA clones of RNA viruses, can be developed and used for genetic cell-based screening assays. For example, two kinds of reverse genetics systems, full-length infectious cDNA clones and replicons, have been developed for a number of flaviviruses (A. A. Khromykh, et al., J. Virol. (1997) 71:1497-1505; M. S. Campbell, et al., Virol. (2000) 269:225-237; R. J. Hurrelbrink, et al., J. Gen. Virol. (1999) 80:3115-3125; M. Kapoor, et al., Gene (1995) 162:175-180; A. A. Khromykh et al., J. Virol. (1994) 68:4580-4588; C. J. Lai et al., Proc. Natl. Acad. Sci. U.S.A. (1991) 88:5139-5143; C. W. Mandl et al., J. Gen. Virol. (1997) 78:1049-1057; C. M. Rice et al., Science (1985) 229:726-733; H. Sumiyoshi et al., J. Virol. (1992) 66:5425-5431; S. Polo et. al., J. Virol. (1997) 71:5366-5374), including lineage II WNV (V. F. Yamshchikov et al., Virology (2001) 281:294-304). Reporter genes can be engineered into the reverse genetics systems to allow for the monitoring of viral replication levels in the presence of potential inhibitors. However, there have been no reverse genetics systems developed for lineage I WNV strains, which comprise the main pathogen strains involved in human and equine outbreaks, such as the recent 1999 U.S. epidemic. For the purposes of drug screening it would be preferable to use human epidemic-causing lineage I strains for assay setup to ensure that the identified compounds have a direct relevance to human disease. Thus, there is an urgent need in the art for reverse genetics systems based on lineage I WNV for use in the identification of novel anti-WNV chemotherapeutics and vaccines.
Therefore, as detailed above, in addition to the need in the art for novel vaccines and/or anti-flaviviral therapies to immunize against and/or treat infections by WNV and other emerging flaviviruses and members of the flavivirus sero-complex, such as JEV, SLEV, AV, KV, JV, CV, YV, TBEV, DENV-1, DENV-2, DENV-3, DENV-4, YFV and MVEV. There is also an urgent need in the art for new methods of identifying potential chemotherapeutic therapies and vaccines, particularly high-throughput screening assays using genetic approaches to identify inhibitors and vaccines of lineage I WNV and other emerging flaviviruses and members of the flavivirus sero-complex, such as JEV, SLEV, AV, KV, JV, CV, YV, TBEV, DENV-1, DENV-2, DENV-3, DENV-4, YFV and MVEV.